Glioblastoma multiforme (GBM), is the most common and most aggressive malignant primary brain tumor in humans. Despite multimodal aggressive therapy, GBM is fatal?with survival over 3 years being considered long-term. More therapeutic targets and treatment options are clearly needed. In our preliminary study, we identified a novel oncogene, AVIL in GBMs. AVIL gene is overexpressed in all the glioblastoma cell lines, including glioblastoma stem cells, and clinical cases, but is hardly detectable in non-cancer astrocytes, neural stem cells, and brain tissues. Silencing AVIL resulted in complete eradication in GBM cell cultures, but had little effect on the astrocyte control cells. In animal models, silencing AVIL dramatically inhibited in vivo xenografts in mice. Conversely, overexpressing AVIL in cell culture systems promoted tumorigenesis, enabled fibroblasts to escape contact inhibition, and transformed immortalized astrocytes. In patient cohorts, higher expression levels of AVIL in gliomas correlated with worse prognosis. Most importantly, GBM cells are more susceptible to small molecule inhibitors of AVIL. These evidences support the premise that AVIL is an Achilles heel of GBMs, targeting which may be an effective approach for GBMs. In this application, we propose the systematic investigation of AVIL as a novel target against GBMs. Aim 1: Determine the efficacy of targeting AVIL in vitro and test AVIL expression as a biomarker of sensitivity. We have shown the critical role of AVIL in some GBM and GSC cell lines. Here, we will investigate its role in additional glioblastoma lines, GSCs, and short-term explants of GBM patient derived xenograft cultures and potential synergistic effect with CDK4/MDM2. We will test the efficacy of tet-inducible shRNAs targeting AVIL, and small molecule compounds we identified through small molecule screen. In addition, we will determine whether AVIL expression serves as a biomarker for sensitivity to AVIL inhibition. Aim 2: Determine the efficacy of targeting AVIL in animal GBM models. We will use shRNA and small-molecule inhibitors in xenograft models to test both the efficacy and safety of targeting AVIL in vivo. In addition, we will use Avil knockout mouse model by crossing them with glioma mouse models to test whether Avil expression is necessary for glioma tumorigenesis in mouse. Aim 3: Investigate the downstream targets and mechanisms of AVIL inhibition and identify potential pharmacodynamic biomarkers. We have preliminary data suggesting AVIL regulates LIN28B, and FOXM1 may mediate the effect in U87 cells. We will investigate whether LIN28B and FOXM1 are functionally critical targets of AVIL, and whether they are potential pharmacodynamic biomarkers to evaluate treatment efficacy of AVIL-targeting therapy. Aim 4: Investigate potential resistance mechanisms of targeting AVIL. The goal is to anticipate challenges to AVIL-based treatment before they arise in the clinical setting. The proposed study will have a significant impact on the understanding and treatment of GBMs. The findings will pave ways to target AVIL as a novel oncogene, and lead to the development of novel therapeutic approaches for the deadly disease.